1. Field of the Invention
The present invention relates to a method for discriminating and counting erythroblasts. In particular, the present invention relates to a method for accurately discriminating and counting erythroblasts by flowcytometry.
2. Description of the Prior Art
The discrimination and counting of erythroblasts are of great use in a field of clinical examination since it provides very useful information for the diagnosis of diseases and prognosis of diseases.
Erythroblasts, which are also called nucleated red blood cells, are normally contained in the bone marrow, but not in peripheral blood except newborns. The appearance of erythroblasts in peripheral blood indicates a possible presence of a disease, such as leukemias, hemolytic anemia, iron deficiency anemia or pernicious anemia, and other non-hematologic/oncologic disorders. Thus the discrimination and counting of erythroblasts are very effective for the diagnosis of any such disease and for the prognosis in some of them.
Conventionally, it has been usual to prepare a smear of blood, add an appropriate dye to the smear of blood and examine it by a microscope for discriminating and counting erythroblasts. However, such a method requires not only time-consuming, complicated pretreatment of blood for examination but also considerable expertise and skills for obtaining accurate results.
Recently, there have been available a variety of apparatuses for the fully automated discrimination and counting of leukocytes based on the principle of a flowcytometer, and there have been proposed a number of methods for analyzing blood contents using such apparatuses.
For example, Japanese Unexamined Patent Publication No. HEI 4(1992)-268453 discloses a method for discriminating and counting erythroblasts in a blood sample. This method includes the treatment of the blood sample with an acid reagent fluid of hypotonic osmolarity, the staining of nuclei of erythroblasts with a fluorescent dye solution and the detection of scattered light and fluorescent light by a flowcytometer.
Japanese Unexamined Patent Publication No. HEI 5(1993)-34251 discloses a method of determining erythroblasts. This method includes the treatment of a blood sample with an acid reagent fluid of hypotonic osmolarity, the staining of the blood sample with four kinds of dyes including Astrazon Yellow 3G and Neutral Red which are fluorescent dyes and the detection of red fluorescent light and green fluorescent light by a flowcytometer.
Published Japanese translation of PCT international publication for patent application No. HEI 8(1996)-507147 discloses a method for determining nucleated red blood cells by detecting forward scattered light or fluorescence-side scattered light by a flowcytometer using a specific amount of a non-quaternary ammonium salt, an aliphatic aldehyde, a non-phosphate buffer, a reagent having a specific pH and a specific osmolarity and a nuclear dye such as ethidium homodimer.
U.S. Pat. No. 5,559,037 discloses a method for counting erythroblasts. This method includes the lysis of erythrocytes and cell membranes of erythroblasts, the staining with a vital nuclear dye capable of staining erythroblasts but not leukocytes and the detection of scattered light at two different angles and fluorescent light by a flowcytometer.
In these methods, however, cell membranes of leukocytes as well as erythroblasts become easily damaged especially in a hematologic sample with increasing time after collection of blood. Accordingly, some leukocytes are stained with the dye that on purpose to stain erythroblasts. As a result, erythroblasts cannot be accurately determined. For example, in the case where scattered light and fluorescent light are detected, a site where erythroblasts appear overlaps or overlays a site where leukocytes appear. Especially, when lymphoid cells are damaged, it becomes more difficult to distinguish erythroblasts from damaged lymphoid cells clearly and thus the presence of erythroblasts cannot be detected accurately.
Moreover, in recent years, more and more medical laboratories send blood samples taken from patients to institutions specialized for mass examination in order to reduce costs and improve efficiency. In such a case, it sometimes takes one day or more from the collection of blood to the examination.
Besides, it is difficult to accurately discriminate and count erythroblasts in a sample containing lymphoblasts or a sample in which chemotherapy or the like has made membranes of leukocytic cells ready to be damaged by an hemolytic agent, even if the sample does not go through change with time.
Additionally, Japanese Patent Publication No. HEI 8(1996)-1434 discloses a method for identifying nucleated red blood cells and the like. This method includes the addition of thiazole orange to a blood sample, the addition of two kinds of fluorescent labeled antibodies, anti-CD45 and anti-CD71, and the detection of signals at at least three fluorescent channels and at least two light scattering channels by a flowcytometer.
This method uses two antibodies and one fluorescent dye as reagents and has a disadvantage in that the reagents are expensive. Since the method can examine immature, nucleated red blood cells only with the combination of these specific antibodies with the specific dye, it is impossible to analyze erythroblasts cheaply by use of this method.
Further, Japanese Unexamined Patent Publication No. HEI 2(1990)-73157 discloses a method for analyzing various kinds of cells including nucleated red blood cells by detecting signals at least three fluorescent channels and at least two light scattering channels by a flowcytometer using two kinds of fluorescent nucleic acid dyes and a fluorescent monoclonal antibody.
According to this method, in order to discriminate erythroblasts from leukocytes, a blood sample is stained with the fluorescent monoclonal antibody, and side scattered light is measured. However, since this publication lacks description about the distinction of erythroblasts from platelets and debris, it is difficult to count erythroblasts exactly by this method.
Japanese Patent No. 2620810 discloses a method for detecting fluorescent light and scattered light by a flowcytometer. The method includes the lysis of erythrocytes and the addition of a monoclonal antibody, a fixative and a nucleic acid dye which binds with DNA first.
According to this method, since a sample must be first subjected to erythrolytic treatment, the sample must be subjected to centrifugal cleaning immediately after the erythrolytic treatment. Thus absolute counting is difficult. Moreover, since this centrifugal cleaning involves complicated operation, results of detection vary significantly depending on the skill of an examiner.
Under these circumstances, there has been a demand for an easy, inexpensive method for analyzing erythroblasts accurately even in a hematologic sample collected some time before. There has also been a demand for a method for classifying and counting erythroblasts according to a degree of maturity thereof. However, such method has not been established so far.